He blot was developed by use of the SuperSignal Ultra chemiluminescence detection system (Pierce) and recorded by the use of Hyperfilm ECL (Amersham Biosciences).Glucose uptake assay Glucose uptake rates were measured by assessing the incorporation of D-[U-14C] glucose [25060 mCi (9.253.33 GBq)/mmol; Perkin Elmer Life Sciences] into germinating conidia. Briefly, 1.209 conidia was inoculated into 300 ml MM containing 2 glucose (w/v) as a carbon source, and incubated for 6 hr at 37in an orbital shaker (180 rpm). Germinating conidia were harvest by centrifugation (3000 rpm) for 5 min and washed four times with ice-cold MM without a carbon source to eliminate traces of glucose. Washed conidia were gently resuspended. For the glucose transport analysis, aliquots of 250 ml (of 2.507 germinating conidia) containing D-glucose (0.120 mM) were dispensed into 2-ml tubes plus 1 ml radiolabeled glucose (0.2 mCi). Incubation times ranged from 30 to 180 sec at 37with shaking. To quench the reaction, 1 ml ice-cold MM without carbon was added. The germinating conidia were washed twice with 1 ml ice-cold MM without carbon and transferred to 8 ml ScintiSafe Econo1 scintillation liquid (Fisher Scientific). The D-[U-14C] glucose taken-up by cells was measured using Tri-Carb 2100TR Liquid Scintillation Counter.Vudalimab ROS detection Intracellular ROS levels were monitored with the oxidant-sensitive probe 5-(and 6)-chloromethyl-29,79-dichlorofluorescin diacetate CMH2DCFDA (Invitrogen). Liquid YUU medium (50 ml) was inoculated with 107 conidia and incubated on a rotatory shaker (180 rpm) for 6 hr at 37 The nonstarved cultures were centrifuged at 4000 rpm and the pellet was resuspended in 2 ml YUU.Luteolin The starved culture was centrifuged at 4000 rpm, the pellets were washed in MM without anyn Table 2 Oxygen consumption in the wild-type and DatmA mutant strain Treatmentsa Respiration medium Antimycin Ab SHAMc TMPD/Ascorbated KCNeaWild-type 43.PMID:36628218 90 6 3.2 30.10 6 1.2 3.25 6 0.3 28.66 6 0.9 2.20 6 0.DatmA 21.40 6 4.06 14.03 6 3.45 2.10 6 1.52 11.85 6 1.50 3.80 6 0.b 0.5 mg Antimycin A. c 2.5 mM SHAM (salicylhydroxamic acid). d eMean 6 SD of three independent experiments. The values represent the rate of oxygen uptake and were expressed as ng atoms O/min/3 07 cells.ml21.150 mM TMPD (N.N.N.N-tetramethyl-p-phenylenediamine) and 0.5 mM ascorbate (ASC). 1.0 mM KCN.Volume 4 January 2014 |ATM Kinase and Carbon Starvation Response |carbon source, centrifuged again, resuspended in MM without a carbon source and incubated for another 1 hr to starve the culture. The starved cultures were centrifuged at 4000 rpm and the pellet was resuspended in 2 ml MM without a carbon source. After starvation, the ROS assay was prepared from 20 ml of either culture (starved or nonstarved) plus 180 ml fresh YUU or MM without a carbon source and 2.9 mg/ml CM-H2DCFDA in a 96-well imaging plates (BD Falcon). The assay plate was incubated at 37for 30 min under shaking. The arbitrary fluorescence units (AFUs) were measured at 503 nm of excitation and 529 nm of emission in the fluorimeter Synergy (Biotek) using the Gen5 software. RNA isolation, cDNA synthesis and quantitative PCRs Mycelia were harvested by filtration, washed with sterile water, immediately frozen in liquid nitrogen and ground into a powder while frozen. Total RNA was extracted with Trizol (Life Technologies) and RNAse-free DNAse treated as described by Semighini et al. (2002) and then purified using the RNeasy Plant Mini Kit (Qiagen). RNA integ.